Project 3: Biological effects of death-regulatory genes in ischemia In Project 3 we will directly test the hypothesis that the death of some neurons after ischemia results from the induced expression of specific death-promoter genes, while the survival of other neurons is promoted by the expression of death-suppressor genes. In Project 1, we have used the regional expression of known death-promoter and death-suppressor genes after ischemic injury to vulnerable or resistant brain regions to identify those genes which are likely to play a role in the survival or death of ischemic neurons; we have used subtractive hybridization to discover novel genes that may play a similar role. In Project 2 these neurons and novel candidate genes will be characterized in culture models of neuronal death. In order to directly test the role of these identified gene products in ischemic cell death, we will block expression of candidate death-promoter or death-suppressor genes in vivo using antisense oligonucleotides. Alternately, we will express candidate death-promoter or death-suppressor genes in vivo using a recombinant HSV-based vector. The effect of altered gene expression on cell survival following transient global ischemia and in ischemic tolerance (a model in which brief sub lethal focal ischemia protects neurons from subsequent prolonged ischemia) will be assessed histologically. The role of a putative death-promoter gene will be supported if blocking its expression increases cell survival after ischemia while overexpression increases the vulnerability of hippocampal neurons to ischemia. The role of a putative death-suppressor will be supported if blocking its expression blocks ischemic tolerance while overexpression reduces cell death following ischemia. The results of the studies in Project 3 will allow us to confirm the role of specific genes in ischemic cell death in vivo; this is a critical step in identifying candidate targets for novel therapies to reduce infarct size in stroke.